This Exploratory/Developmental project combines the resources and skills of laboratories at Cornell University and the University of Florida in studies to define the antigenic structures of the capsids of 3 different serotypes of adeno-associated virus (AAV). We would use the information obtained to prepare variant viruses with improved antigenic properties for applications in corrective human gene therapy as well as to understand the general properties of antibody binding and neutralization of viruses by antibodies. Although the high resolution structures of several AAV capsids are now available, there is still little information about their general or specific antigenic structures. The overall goals of this project are therefore to define the structural bases of antibody-capsid interactions using mouse IgG or IgM monoclonal antibodies (MAb) to the AAV1, AAV2, and AAV5 capsids, which are representative of the antigenic range of AAV serotypes in humans. We would also isolate clones of human antibody sequences that recognize the capsids by screening a yeast expression library of human antibody VH and VL sequences. We have already prepared panels of MAb against AAV1 and AAV5 capsids which will be tested as IgGs or Fabs for binding and neutralization of the various viruses. In addition, a number of AAV2 antibodies are already available. Antibodies or antibody domains will be used to select escape mutants, and the sequence variation observed will be used to define properties of the epitopes on the capsid surfaces. Cryo-electron microscopy and image reconstruction of Fab/capsid complexes will also be used to define to moderately high resolution the interactions of several antibodies with the capsid surfaces. These data will be used to define the dominant immunogenic structures on the native capsids, to determine if the capsids utilize common epitopes, and will allow comparison of the results obtained with each of the methods. The combined information will used to guide the preparation of capsid mutants designed to avoid binding or neutralization by most of the antibodies to the capsids, while retaining normal infectivity and cell or tissue tropisms. Mutant capsid genes would be used to generate AAV vectors which would be tested for binding to and neutralization by the various IgG and IgM MAbs and also by polyclonal antibodies. These capsids would also be tested for their specific receptor binding and transduction/tropism properties. Public health significance: Antibodies are central defenses against all viruses of vertebrates, but questions remain about the structural bases of antibody binding to the capsids, the bases of antigenic diversity and variation, as well as the mechanisms of neutralization. The many serotypes of AAV are being widely used in unmodified or modified forms as vectors with distinct properties; however many people carry neutralizing antibodies against various serotypes, and the use of AAV vectors can lead to the development of antibody immunity. In this study we seek to define the antigenic structures if the viruses so that the adverse effects of antibodies on gene therapy trials can be better understood, and perhaps avoided in the future. [unreadable] [unreadable] [unreadable]